Amino acid chalcogen analogues as tools in peptide and protein research

The chalcogen elements oxygen, sulfur, and selenium are essential constituents of side chain functions of natural amino acids. Conversely, no structural and biological function has been discovered so far for the heavier and more metallic tellurium element. In the methionine series, only the sulfur‐containing methionine is a proteinogenic amino acid, while selenomethionine and telluromethionine are natural amino acids that are incorporated into proteins most probably because of the tolerance of the methionyl‐tRNA synthetase; so far, methoxinine the oxygen analogue has not been discovered in natural compounds. Similarly, the chalcogen analogues of tryptophan and phenylalanine in which the benzene ring has been replaced by the largely isosteric thiophene, selenophene, and more recently, even tellurophene are fully synthetic mimics that are incorporated with more or less efficiency into proteins via the related tryptophanyl‐ and phenylalanyl‐tRNA synthetases, respectively. In the serine/cysteine series, also selenocysteine is a proteinogenic amino acid that is inserted into proteins by a special translation mechanism, while the tellurocysteine is again most probably incorporated into proteins by the tolerance of the cysteinyl‐tRNA synthetase. For research purposes, all of these natural and synthetic chalcogen amino acids have been extensively applied in peptide and protein research to exploit their different physicochemical properties for modulating structural and functional properties in synthetic peptides and rDNA expressed proteins as discussed in the following review.


| INTRODUCTION
In early days of X-ray crystallography of proteins, one of the main difficulties was the trial and error soaking procedure for the production of heavy atom derivatives. It was with the fast and efficient advances in rDNA technology that using methionine-auxotrophic Escherichia coli strains quantitative biosynthetic replacement of methionine by selenomethionine was used by Hendrickson et al 1 to generate atomic mutants of proteins as a new approach to solve the phase problem in protein crystallography with the multiwavelength anomalous dispersion method. 2 This new procedure led to increasing interest in chalcogen analogues of amino acids in the methionine and serine/cysteine series as reviewed in previous literature. 3,4 These analogues are shown in Figure 1 and differ in their physicochemical properties such as electronegativity, atom volume, and carbon-metal bond length depending upon the chalcogen atoms in their side chains.

| CHALCOGEN ANALOGUES OF METHIONINE
In the methionine series, only methionine is a proteinogenic amino acid, whereas selenomethionine and telluromethionine are natural amino acids that are inserted into natural proteins because of the tolerance of the methionyl-tRNA synthetase. [10][11][12]

| Selenomethionine and telluromethionine protein variants for X-ray crystallography
Intensive optimization of the expression conditions in Escherichia coli was required for quantitative replacement of methionine in proteins with the related chalcogen analogues to produce the highly isomorphous protein variants. Moreover, it was noticed that the proteins have to be biosynthesized in the folded form since oxidation of the selenium and even more of the tellurium occurs in the unfolded states as exists in inclusion bodies. 3,[13][14][15][16] The chalcogen protein analogues are isomorphous and can be considered as atomic mutants of the native methionine proteins. Therefore, no significant changes in the overall shape, steric complementarity, and occupation volume are gen-FIGURE 1 Chalcogen amino acids discovered in natural compounds except methoxinine FIGURE 2 Isosteric synthetic chalcogen analogues of tryptophan and phenylalanine erated. Minor steric effects cannot be excluded as shown by the electron difference maps ( Figure 3), but the protein folds show generally sufficient plasticity to accommodate such minor differences even in the case of tellurium. On the other hand, the telluro-analogues provide directly heavy atom derivatives for the classical multiple isomorphous replacement method, but the production is more difficult. Conversely, the seleno-mutants in most cases are not suitable for this method and are more appropriate for multiwavelength anomalous dispersion experiments that require, however, synchrotron radiation with precise beam wavelength control.

| Methoxinine as methionine-oxide mimic
This additional chalcogen analogue of methionine with sulfur replaced by oxygen differs from methionine particularly because of its strong hydrophilicity compared with methionine, selenomethionine, and telluromethionine. With this hydrophilic property, it resembles the methionine-oxide. Because of this particular property, it was reasonable to expect that a related prion protein (hrPrP- ) Moxanalogue could be useful for analysis of the proposed but still not clearly documented hypothetical role of Met oxidation as the main cause of the prion protein conversion to its scrapie form. 17,18 Indeed particularly Met-205 and Met-206 oxidation was suggested to be responsible for the conformational transition of the α-helix into βsheet and thus for the aggregation and the neurotoxic amyloid formation (see Figure 4). 20 The in vitro aggregation propensity of the Mox-hrPrP c -  analogue was found to be significantly increased compared with that of the native Met-protein ( Figure 5). 19 Even the content of α-helix as derived from the circular dichroism (CD) spectrum was visibly decreased with concomitant shift to βsheet. With the large number of methionine residues in the PrP-  protein, it is difficult to confirm that the oxidation of a few selected methionine residues initiates the conformational transition. However, this is not only supported by the results obtained with the Mox-rhPrP c -  analogue but also by experimental results obtained with model peptide of Dado and Gellman 22 where a significant conformational shift from α-helix to β-sheet could be observed by replacing the hydrophobic norleucine residues as methionine analogues with the hydrophilic Mox residues. 19
Tryptophan represents a suitable target for its replacement in proteins with synthetic isosteric analogues of near-equal volumes and approximately identical electron densities. Moreover, Trp is known to occur rather rarely in proteins as it represents only about 1% of all residues of globular proteins. 24 Correspondingly, chalcogencontaining Trp analogues provide quasi-site-specific probes for studying protein structure, dynamics, and function via their incorporation into recombinant proteins in response to the Trp UGG codons by fermentation in Trp-auxotrophic E coli host strains and applying the selective pressure incorporation (SPI) method. [25][26][27] Under routine bioexpression protocols, an almost quantitative incorporation of both β-(thienopyrrolyl)alanines in annexin V that contains only one Trp residue and in the pseudo-wild-type barstar mutant C40A/C82A/P27A/ W38F (b*) with two Trp residues was achieved. 28 The barstar mutant is obtained in inclusion bodies and is refolded into the native form during the purification procedure. Both the annexin V and barstar Trp analogues proved to be stable, and despite exposure to air and light for days, a degradation of the thienopyrrolyl moieties was not observed. Both proteins were found to exhibit reduced thermal stability but full retention of the biological activities. Although these protein mutants retain the secondary structure of the native proteins, they were found to differ significantly in optical and thermodynamic properties.
In view of these positive results, the β-selenolo [3,2-b]pyrrolyl-Lalanine was incorporated into human annexin V and barstar mutant b* C40A/C82A/P27A as model proteins in Trp-auxotrophic E coli using the SPI method. The seleno-proteins were obtained in yields comparable with those of the wild-type proteins and the β-(thienopyrrolyl)-L-alanine mutants. 29 Both selenium-containing mutants were found to exhibit crystallographic isomorphism with the parent proteins. Although the selenium-containing Trp analogue replaces in annexin V the fully exposed Trp-187 and in the barstar mutant the two Trp residues 44 and 38 partially or fully exposed to solvent, their exceptional stability can be regarded as a considerable advantage over selenomethionine in replacement experiments for crystallographic phasing. The most probable explanation for this observed high stability is a resonance stabilization of the Se atoms in aromatic rings where the chalcogen atom is less prone to oxidation than in dialkylselenides, such as selenomethionine. Moreover, in cases where Sem is less suitable for diffraction analysis because of the atomic mobility factors, the use of the selenotryptophan analogue could be an alternative.

| SULFUR-, SELENIUM-, AND TELLURIUM-CONTAINING PHENYLALANINE ANALOGUES
On the basis of early reports of du Vigneaud et al 30 on the incorporation of β-2-thienylalanine (5 in Figure 2) into proteins, already in the early 1960s, it could be demonstrated that this phenylalanine analogue is inserted into the enzyme β-galactosidase in a Phe-requiring auxotroph of E coli. 31 This result confirms the largely isosteric character of the thiophene ring with the benzene ring although of less aromatic character, having a resonance energy of 25 kcal/mol as compared with 36 kcal/mol for benzene. The catalytic properties of the enzyme with about 95% of the phenylalanine residues replaced by the analogue were retained; however, the enzyme was found to be significantly less stable to heat, urea, and enzymatic degradation.
Later in the 1970s, the β-2-thienylalanine has also been incorporated synthetically in various peptide hormones such as vasopressin and bradykinin leading to full retention or even increased bioactivities. 32,33 Conversely, the β-2and β-3-selenienyl-L-alanines (6 and 8 in Figure 2) have been synthesized as potential diagnostic agents for disorders in the pancreas 8 but so far did not find any application in replacement experiments for crystallographic phasing. However, very recently, a detailed study on the stability of various organotellurium compounds revealed for 2-alkyl-tellurophenes high stability to aerobic oxidation in both organic and aqueous solutions and a limited toxicity in cell-based assays (IC 50 ≥ 200μM) 34 making such tellurophenes ideal probes for mass cytometry. Correspondingly, β-2-tellurienyl-L-alanine was synthesized knowing that the related sulfur-and seleniumcontaining phenylalanine mimics are efficiently incorporated into proteins because of the tolerance of the phenylalanine-tRNA synthetase. 9 The telluro-phenylalanine analogue was found to behave as an excellent phenylalanine isostere since it is incorporated into proteins via the native translation machinery without phenylalanine starvation in vitro and in vivo allowing its measurements by mass cytometry techniques. It looks also as a promising probe in X-ray crystallography as the electron density of tellurium is known to generate isomorphous and anomalous difference Patterson maps. In this context, its properties should be superior to those of telluromethionine that suffers from a more facile oxidation when exposed on the protein surface (see Section 2.1) but with the drawback of a generally higher number of Phe residues in proteins compared with Met.

| SERINE AND CYSTEINE CHALCOGEN ANALOGUES
In the serine/cysteine series, also selenocysteine is a proteinogenic amino acid that is inserted into proteins by a special translation mechanism, 35,36 while the tellurocysteine is again only tolerated by the cysteinyl-tRNA synthetase. 11 In this series of chalcogen amino acids, the related hydroxyl, thiol, and selenol side chain groups of serine (pK a 13), cysteine (pK a 8.25), and selenocysteine (pK a 5.24-5.63) differ significantly in the nucleophilicity leading to different structural and functional properties. [37][38][39] While the cysteine residues form the known important structural disulfide crosslinks for stabilization of tertiary structures in peptides and proteins, 40,41 the peroxides of serine would be biologically too dangerous because of their oxidation power. Although serine and cysteine differ considerably in the nucleophilicity, both residues play a central role in proteases. In the catalytic triad of serine proteases, an aspartyl residue is required to increase via hydrogen bonding the nucleophilicity of the serine hydroxyl group, while such a helping residue is not required in the cysteine proteases.
Besides the established role of disulfides crosslinks in cysteinerich peptides and proteins for stabilizing related tertiary structures, vicinal disulfides in specific sequence motifs such as the Cys-Xaa-Yaa-Cys act as highly efficient active sites of thiol-protein oxidoreductases. 42,43 This active-site motif of thioredoxins and glutaredoxins is involved in the reduction of intermolecular and intramolecular disulfide bonds and other forms of oxidized cysteines, and in protein disulfide isomerases, this bis-cysteinyl motif catalyzes the oxidative folding of secretory proteins into their native structures. 44 This has, therefore, been called a "rheostat at the active center." Indeed, changes of the residues that separate the two cysteines affect the redox potentials, thus structuring the proteins for a particular redox function. 45,46 Besides the effect of the dipeptide intervening sequence, 47 which is also well supported by the redox potentials of synthetic fragments related to their active sites 48 (   Table 1), the redox properties of these active sites are strongly governed by the conformational restrictions imparted by the overall almost identical thioredoxin-like structure of the oxidoreductases. 42,47 These structural effects were further evidenced by the redox potentials of conformationally restricted cyclic active-site (bis-cysteinyl)hexapeptides related to oxidoreductases, 56 when compared with those of the linear unconstrained peptides (Table 1).

| Selenocysteine as isosteric replacement of cysteine
Selenium and sulfur possess very similar atomic sizes, bond lengths to carbon, homodimeric bond lengths, and electronegativity. Correspondingly, selenocysteine represents an isosteric analogue of cysteine. 3,4,57,58 This was already demonstrated with early synthetic selenocysteine/selenocystine analogues of bioactive peptides such as oxytocin, somatostatin, and rat α-natriuretic peptide, which retained the bioactivities of the parent Cys-peptides. 58 The isosteric character was then fully confirmed by comparing the NMR structures of With the Boc strategy where the selenol function is protected by the methylbenzyl group, the facile deselenization of the Sec (Mob) in the iterative piperidine-mediated Fmoc-deprotection steps is avoided. 68 Moreover, the facile racemization of Sec residues can readily be suppressed by the in situ neutralization protocol of solid-phase peptide synthesis. 66 The final HF deprotection step at 0°C for all protecting groups leads to the Sec residues already oxidized to the expected diselenide or mixed selenosulfide bonds even under the very strong acid conditions. 63,72 Alternatively, a cost-efficient approach can be applied for the synthesis of short selenocystine peptides by reacting the related β-chloroalanyl-peptides with Na 2 Se 2 73 in protic solvents such as methanol avoiding by this way protection and deprotection of the selenocysteine residues. 74 Using prokaryotes incorporation of selenocysteine into recombinant natural Sec-proteins is rather inefficient 75 and requires the Secexpression machinery. 76 Therefore, recombinant expression of natural and designed seleno-proteins is not yet a routinely applied method for the production of seleno-peptides and -proteins. 77  This difference may likely derive from absorption of the selenol on the dropping mercury electrode. Therefore, alternatively, the redox potentials of diselenide and mixed selenide/sulfide were determined for the Sec 11,14and Sec 11 ,Cys 14 -Grx1-10-17 octapeptides related to the active site of glutaredoxin-1 (Grx1) ( Table 2). 92 This choice was based on the observation that this short active-site fragment shows only minimal effects imparted by the 3D fold of the protein on the apparent redox potential of the bis (cysteinyl)-octapeptide Cys 11,14 -Grx1-10-17 (E′ 0 = −215 mV). 48 Moreover, it was only minimally  As shown in Table 2, the redox potential of the bis  (Table 2). 72

| Synthesis of cysteine-rich peptides by the selenocysteine strategy
The strong differences of more than 100 mV between the redox potentials of disulfides and diselenides and to lesser extents between disulfides and selenosulfide groups make formation of a diselenide and even mixed selenosulfide highly favored over that of a disulfide bond.
It was known that oxidative folding of cystine-rich peptides already with two disulfide bonds rarely produces quantitatively the native disulfide isomer like in apamin 95 but more often mixtures of the native disulfide isomer contaminated at differing extents by the nonnative isomer like in endothelin-1. 96 The ability of selenocysteine to act as an internal "chaperone" and thus to dictate the folding pathway of cysteine-rich peptides was first confirmed by the synthesis of seleno-endothelin-1. 59  Sec-analogues in rather high yields of 61%, 42%, and 47%, respectively. 103 Since enhanced kinetics of disulfide formation was observed in selenopeptides, the rate increase has been in first instance attributed to conformational effects. However, diselenides as additives were known to efficiently catalyze oxidative protein folding. 104,105 Correspondingly, the kinetics of oxidative folding of the μ-conotoxin SIIIA and ω-conotoxin GVIA, both containing the cystine knot ICK, were compared with the kinetics of formation of the correctly folded [Sec [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] ]-SIIIA and [Sec [8][9][10][11][12][13][14][15][16][17][18][19] ]-GVIA. The results clearly revealed an efficient catalytic effect of even intramolecular diselenides in the formation of the correct disulfides. 106 This can be explained by the fact that the diselenide bond is weaker than the corresponding disulfide bond and thus is easier to break and to be cleaved by thiols. The way that selenium accelerates in the case of a thiol/diselenide exchange reaction is that the electrophilic selenium atom being attacked is a much better electrophile than sulfur and accelerates the reaction rate 100-fold more in comparison with sulfur. 107 (Figure 8). 101,102 This surprising strong effect of intramolecular catalysis of diselenide groups on the folding of cysteine-rich peptides was compelling to apply the selenocysteine strategy in the attempt to advance in the still existing problem of efficient insulin syntheses as recently reviewed. 109  was the "Ester-Insulin" of Kent and associates in which the crosslinking of the A-and B-chain is achieved by esterification of the β-hydroxyl group of Thr B30 with the γ-carboxyl group of Glu A4 yielding an insulin precursor of a foldability similar to that of natural proinsulin; a simple saponification of the ester group produces the insulin in correctly folded structure and yields similar to those obtained with optimized single-chain proinsulins (Figure 9). [111][112][113] This experimental evidence clearly confirms that a simple crosslinking of the A-and B-chain to the heterodimer via an ester linkage would suffice for reducing the entropic penalty in the oxidative folding of the two chains in a manner similar to the proinsulin precursor. These findings were strongly suggesting an application of the selenocystine strategy to produce an A7-B7 or A20-B19 diselenide as interchain crosslink with similar atom economy as the ester-insulin but with the advantage of the intramolecular folding catalysis by the diselenide (Figure 10). The synthetic bovine seleno-insulin was found to exhibit the identical tertiary structure as the wt insulin, practically the identical bioactivities in terms of phosphorylation levels of Akt and GSK3β in Hela cells, but significantly increased resistance to the insulin degrading enzyme, a very positive property for a fully active insulin analogue. 114 The highly beneficial effect of the catalysis by the intermolecular diselenide and the significant reduction of the entropic penalty by the highly favored interchain-diselenide crosslink was fully confirmed.

FIGURE 10
Possible placement of the two selenocysteines in synthetic linear A-and Bchains for induction of diselenidecrossbridged heterodimeric insulin-folding precursors

FIGURE 9
Conversion of ester-insulin to Lys B28 ,Pro B29 -insulin variant via oxidative folding and saponification according to Kent and associates 111,112 From extensive studies on folding of insulin and proinsulin, it was known that the intrachain disulfide Cys A6 -Cys A11 is important for induction of the overall insulin structure, even if not essential. 115 However, this disulfide if preferentially installed leads to conformational constraints that structurally preorganize the A-chain facilitating the correct formation of the remaining interchain disulfides. 116 On

| Tellurocysteine as isosteric replacement of cysteine
Unlike the chalcogens oxygen, sulfur, and selenium that find biological use, for tellurium, no biological function is known so far. However, in rare cases, bacteria and fungi grown in the presence of sodium tellurite and in the absence of sulfur were found to produce telluriumcontaining amino acids (tellurocysteine, tellurocystine, and telluromethionine) and to bioincorporate these into proteins, 11,121 a process that has to occur by the tolerance of the methionyl-and cysteinyl-tRNA synthetases. On the basis of experience with the accumulation of selenium in proteins of Spirulina platensis, a blue-green algae, in the form of selenomethionine and selenocysteine, 122,123 accumulation of tellurium into the phycobiliproteins phycocyanin and allophycocyanin of S platensis was found to proceed successfully leading to enhanced antioxidant activities of both proteins. 124 These results strongly support such novel organic tellurium species for application in antioxidation for the treatment of diseases related to oxidative stress.
On the basis of these results, even recombinant expression of the tellurocysteine-containing glutathione-S-transferase from Lucilia cuprina (LuGST-1) was found to occur in good yields with a Cysauxotrophic expression system in the presence of synthetic tellurocysteine, 125 although tellurocysteine is apparently on the borderline in terms of the substrate tolerance of cysteinyl-tRNA from E coli. 126 Alternatively, the Tec amino acid was inserted into subtilisin to form semisynthetic tellurosubtilisin 127,128 ; however, this chemical modification has the disadvantage that it can be applied only at active-site serine residues like Ser 221 in subtilisin. Thus, the development of a general strategy for the incorporation of Tec into proteins so far remains a challenging task.
On the other side, the redox potential of tellurocysteine/ tellurocystine determined by cyclic voltammography using mercury film coated glass carbon electrode is characterized by an intrinsically lower redox potential (−850 mV versus Ag/AgCl) than that of selenocysteine/selenocystine (−640 mV versus Ag/AgCl) making it an interesting mutant of Cys-and Sec-containing proteins because of its considerably more reducing properties. 126 The synthesis of tellurocysteine has been reported 125,129 as well as the synthesis of (Boc-Tec-OH) 2 130 and H-Tec (Meb)-OH 129 as potential intermediates for the synthesis of Tec-peptides. However, oxytocin as the only reported synthetic Tec-containing peptide so far was prepared according to the synthetic route shown in Figure 13. 129 A comparison of the oxytocin ditelluride with its wild-type and diselenide variant clearly revealed a visibly reduced receptor affinity and functional property (Table 3). 131 However, it was found to be surprisingly stable in view of general observations with organotellurium compounds where the ditelluride group was found to be light-sensitive with tendency to hydrolyze and to decompose forming telluroxides, tellurite, tellurate, and even elemental tellurium. 132 In addition, also the carbon-Te bond (around 200 kJ mol −1 ) is less stable than the carbon-Se bond (234 kJ mol −1 ) and the carbon-S bond (272 kJ mol −1 ) with the carbon-O bond being the most stable (358 kJ mol −1 ). In this context, the Te-alkyl compounds were found to be generally less stable than the Te-aryl or Te-alkylaryl compounds, a fact that was confirmed in the case of telluromethionine-protein analogues (see Section 2.1 and Besse et al 3 ) in comparison with the stability of simple organotellurium compounds. 132 The higher stability of the alkylaryl tellurides could well be exploited for the synthesis of Tec-peptides with Tec (Mob) or Tec (Meb) protected tellurocysteine.
The promising therapeutic properties already reported for simple organotellurium derivatives with antimicrobial, antioxidant, immunomodulatory, and anticancer activities should foster intense research also with small purposely designed Tec-containing low-mass peptides and related derivatives.

| CONCLUSION AND REMARKS
As among the members of the chalcogen elements, oxygen, sulfur, and selenium play vital roles in the chemistry of life, the apparent lack of any biochemical function for tellurium is rather surprising also in view of its properties that are similar to that of sulfur and selenium. While oxygen is essential for life on Earth, sulfur exerts crucial roles in cellular processes, biocatalysis, and protein structures. This may well be the reason why great attention was paid to study and exploit the biochemistry and properties of sulfur as well as more recently of selenium, the latter because of its two faces as it is both toxic to all organisms, but also essential to many bacteria and animal species. 133,134 Thereby, the redox chemistry was found to represent the largest difference between the two chalcogens, a fact that has been exploited in nature to enhance and adjust redox properties of selenium-containing enzymes and proteins, but also in the laboratory to direct oxidative folding of cysteine-rich peptides and enhance their thermodynamic stability in view of potential therapeutic applications (see Section 5). On the basis of the superb results obtained by replacing sulfur with selenium particularly in methionine, but also in cysteine for producing heavy atom analogues with considerable advantages in protein and peptide X-ray crystallography, a similar approach has been proposed also for bioincorporation of telluromethionine (see Section 2.1). This approach has not found the due attention particularly because of the facile oxidation and decomposition of the telluromethionine. Such drawback can, however, be overcome by using the Te-containing phenylalanine and tryptophan analogues.
Indeed, on the basis of optimized synthesis of β-selenolo [3-2b] pyrrolyl-L-alanine and β-selenolo [2-3b]  conversion to the Te-Trp analogue has been reported. 137 Such a tellurium-containing Trp analogue as well as the β-2-tellurienyl-L-alanine as Phe analogue 9 where in both cases the tellurium is incorporated into the aromatic ring system should be significantly more stable to oxidation and decomposition than the alky-Te-alkyl compounds like telluromethionine. 34,130 This observation may well help raising the pharmacological perspective of tellurium compounds even at the level of peptides and proteins from a forgotten to an emerging new chalcogen-containing class of compounds with promising bioactivities.